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Code availability

Data availability

The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD009281, and are presented in Supplementary Table 1. Unprocessed images of all gels and blots (Figs. 1c,d and 5a,c and Supplementary Fig. 2h) are provided in Supplementary Fig. 5. Source data for all graphs are provided in Supplementary Table 2. All other data supporting the findings of this study are available from the corresponding authors on reasonable request.

Acknowledgements

We thank J. Lukas for commenting on the manuscript, researchers at the Groth laboratory for fruitful discussions, R. Baer for sharing the BARD1 plasmid and BARD1-null mouse mammary carcinoma cells, M. Kanemaki for the OsTIR1 antibody, Y. Antoku for assistance with microscopy, and J. V. Johansen for help with statistical analysis. J.R.B. is funded by a Cancer Research UK Career Development Grant (C52690/A19270). Funding for T.B. was provided by the Medical Research Council (grant number MC_UP_1102/2) and European Research Council (ERC StG number 309952). S.L. was supported by a stipend from the Biotechnology and Biological Sciences Research Council. The Groth laboratory is supported by the Danish Cancer Society, Novo Nordisk Foundation, Lundbeck Foundation, European Research Council (ERC CoG number 724436), Independent Research Fund Denmark and Neye Foundation.

Author information

Author notes

Giulia Saredi

Present address: MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, Sir James Black Centre, University of Dundee, Dundee, UK

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Contributions

K.N., G.S. and A.G. conceived the study. K.N. carried out the functional analysis. G.S. performed pull-downs with endogenous BARD1 and SLF1, and established tools and reagents. J.R.B. generated and carried out experiments with BARD1AID/AID cells. B.M.F. prepared materials for nucleosome pull-downs. N.V.N. prepared materials, carried out SILAC nucleosome pull-down experiments and analysed the data. T.E.B. and L.C.C. performed pull-downs with BARD1 and SLF1 mutants. P.A.F. carried out mass spectrometry measurements and analysed the mass spectrometry data. S.L. performed bioinformatics analyses of the mass spectrometry data. T.F. performed structural modelling. J.R.C., T.B. and A.G. supervised the project and analysed the data. G.S. and A.G. wrote the manuscript with input from all authors.

Competing interests

G.S. and A.G. are inventors on a filed patent application covering the therapeutic targeting of ARD interactions with H4K20me0 for cancer therapy. A.G. and T.F. are co-founders of Ankrin Therapeutics.

a, b, Model of the BARD1 ARD structure (green) in complex with histone H4 (L22-K12) tail (yellow) refined by the Rosetta macromolecular modelling package using the 2.4 Å high-resolution crystal structure of TONSL ARD/H4K20me07 (PDBID 5ja4) as template (see method section for further details). The C-alpha Root-Mean-Square Deviation (RMSD) between the BARD1 ARD model in complex with H3-H4 and the BARD1 ARD crystal structure18 (PDBID 3c5r - in absence of H4) is 0.7 Å. In comparison, the main chain RMSD between the ARD domains of the crystal structure of BARD1 (PDBID 3c5r) and TONSL (PDBID rja4) is 0.9 Å. The hydrogen bonding network of predicted key interactions between the positively charged side chains of H18 and K20 (yellow sticks) and the negatively charged E429, D458, E467 and D500 (green sticks) are illustrated by yellow dashed lines. (b) Structural super positioning of BARD1 ARD/H4 (L22-K12) (green/yellow) with the crystal structure (PDBID 5ja4) of TONSL ARD/H4K20me0 (grey). Repositioning of R17 in H4 is indicated by the dashed black arrow. In the TONSL crystal structure R17 makes interactions with both the main and side chain carbonyl of N571. In the model of the BARD1 ARD in complex with H4, NH2 in the amide side chain of the nearby N504 (which is C608 in TONSL ARD) makes an intramolecular hydrogen bond to the backbone carbonyl of N470 resulting in a relocation of R17. The carbonyl oxygen of the N504 side chain is predicted to make a hydrogen bond interaction with the NH amide backbone atom of K16. c, d, Analysis of BARD1 ARD single amino acid mutants in U-2-OS cells induced to express Flag-HA-BARD1 WT or the indicated mutants. Mean with S.D., n = 3. (c) Total protein levels and chromatin-binding were analysed by high content microscopy in cells either directly fixed or pre-extracted before being processed for HA immunofluorescence. (d) High content microscopy of Flag-HA-BARD1 and BRCA1 IR-induced foci analysed 45 min after IR (3.5 Gy). All constructs carried the Q406 sequence divergence, as described in Supplementary Fig. 3d. e, Alignment of the ARDs of TONSL, BARD1 and SLF1 (from H. sapiens, M. musculus and X. tropicalis). The general ARD consensus sequence is indicated above. The arrows indicate the conserved acidic residues.